<Emphasis Type="Italic">Amborella trichopoda</Emphasis>, plasmodesmata,and the evolution of phloem loading

Phloem loading is the process by which photoassimilates synthesized in the mesophyll cells of leaves enter the sieve elements and companion cells of minor veins in preparation for long distance transport to sink organs. Three loading strategies have been described: active loading from the apoplast, passive loading via the symplast, and passive symplastic transfer followed by polymer trapping of raffinose and stachyose. We studied phloem loading in Amborella trichopoda, a premontane shrub that may be sister to all other flowering plants. The minor veins of A. trichopoda contain intermediary cells, indicative of the polymer trap mechanism, forming an arc on the abaxial side and subtending a cluster of ordinary companion cells in the interior of the veins. Intermediary cells are linked to bundle sheath cells by highly abundant plasmodesmata whereas ordinary companion cells have few plasmodesmata, characteristic of phloem that loads from the apoplast. Intermediary cells, ordinary companion cells, and sieve elements form symplastically connected complexes. Leaves provided with ¹⁴CO₂ translocate radiolabeled sucrose, raffinose, and stachyose. Therefore, structural and physiological evidence suggests that both apoplastic and polymer trapping mechanisms of phloem loading operate in A. trichopoda. The evolution of phloem loading strategies is complex and may be difficult to resolve.     

Phloem loading and its development related to plant evolution from trees to herbs

Summary Minor vein structure in various taxonomic groups was described in a previous paper (Gamalei 1989). Here, these results are used to correlate minor vein structure with plant evolutionary, ecological and growth form schemes. The following pattern emerges: reductive evolution from evergreen trees to annual herbs is accompanied by gradually increasing symplastic isolation of the mesophyll and the phloem. This evolutionary tendency is confirmed by the ecological spreading and life-form distribution of modern plants with different types of minor vein structure. The meaning of this phenomenon is discussed. Chilling sensitivity of plasmodesmal translocation is considered to be the main reason. It is suggested that phloem loading for assimilate transport is double-routed. The symplastic route is more ancient and more economical for loading. The apoplastic pathway becomes the main or the only route under unfavorable conditions. The existence of a symplast/apoplast regulatory loading mechanism is suggested. The two loading routes differ in their selectivity for products of photosynthesis which changes their symplast/apoplast ratio which, in turn, determines the composition of the sieve tube exudate. The latter will influence growth and morphogenesis. Correlated changes of structure and function related to photosynthesis, loading, translocation and growth, are analysed with respect to life-form evolution. The influence of the pathway of loading on other processes is discussed.     

The mechanism of phloem loading in rice (Oryza sativa)

Carbohydrates, mainly sucrose, that are synthesized in source organs are transported to sink organs to support growth and development. Phloem loading of sucrose is a crucial step that drives long-distance transport by elevating hydrostatic pressure in the phloem. Three phloem loading strategies have been identified, two active mechanisms, apoplastic loading via sucrose transporters and symplastic polymer trapping, and one passive mechanism. The first two active loading mechanisms require metabolic energy, carbohydrate is loaded into the phloem against a concentration gradient. The passive process, diffusion, involves equilibration of sucrose and other metabolites between cells through plasmodesmata. Many higher plant species including Arabidopsis utilize the active loading mechanisms to increase carbohydrate in the phloem to higher concentrations than that in mesophyll cells. In contrast, recent data revealed that a large number of plants, especially woody species, load sucrose passively by maintaining a high concentration in mesophyll cells. However, it still remains to be determined how the worldwide important cereal crop, rice, loads sucrose into the phloem in source organs. Based on the literature and our results, we propose a potential strategy of phloem loading in rice. Elucidation of the phloem loading mechanism should improve our understanding of rice development and facilitate its manipulation towards the increase of crop productivity.     

Phloem loading. A reevaluation of the relationship between plasmodesmatal frequencies and loading strategies

The incidence of plasmodesmata in the minor vein phloem of leaves varies widely between species. On this basis, two pathways of phloem loading have been proposed: symplastic where frequencies are high, and apoplastic where they are low. However, putative symplastic-loading species fall into at least two categories. In one, the plants translocate raffinose-family oligosaccharides (RFOs). In the other, the primary sugar in the phloem sap is sucrose (Suc). While a thermodynamically feasible mechanism of symplastic loading has been postulated for species that transport RFOs, no such mechanism is known for Suc transporters. We used p-chloromercuribenzenesulfonic acid inhibition of apoplastic loading to distinguish between the two pathways in three species that have abundant minor vein plasmodesmata and are therefore putative symplastic loaders. Clethra barbinervis and Liquidambar styraciflua transport Suc, while Catalpa speciosa transports RFOs. The results indicate that, contrary to the hypothesis that all species with abundant minor vein plasmodesmata load symplastically, C. barbinervis and L. styraciflua load from the apoplast. C. speciosa, being an RFO transporter, loads from the symplast, as expected. Data from these three species, and from the literature, also indicate that plants with abundant plasmodesmata in the minor vein phloem have abundant plasmodesmata between mesophyll cells. Thus, plasmodesmatal frequencies in the minor veins may be a reflection of overall frequencies in the lamina and may have limited relevance to phloem loading. We suggest that symplastic loading is restricted to plants that translocate oligosaccharides larger than Suc, such as RFOs, and that other plants, no matter how many plasmodesmata they have in the minor vein phloem, load via the apoplast.     

A three-step screening procedure to identify the mode of phloem loading in intact leaves

A three-step screening method was developed to identify the mode of phloem loading in intact leaves. Phloem loading of ¹⁴CO₂-derived photosynthate was challenged by p-chloromercuribenzenesulfonic acid (PCMBS) in leaves of dicotyledons with either a symplasmic (type 1, with intermediary cells as companion cells) or apoplasmic (type 2b, with transfer cells as companion cells) minor-vein configuration. Firstly, photosynthate export as the result of phloem loading was measured by collection of phloem exudate from the petiole. The PCMBS had virtually no effect on photosynthate export in representatives of type-1 families (Lamiaceae, Lythraceae, Onagraceae, Saxifragaceae). In contrast, photosynthate export was strongly reduced by PCMBS in representatives of type-2b families (Asteraceae, Balsaminaceae, Dipsacaceae, Linaceae, Tropaeolaceae, Valerianaceae) and type-2b members of polytypical families (Fabaceae, Scrophulariaceae). Secondly, densitometric measurements of leaf autoradiographs demonstrated that the contrast between the mesophyll and the lower-order veins was hardly affected by PCMBS treatment in type-1 species, whereas PCMBS strongly reduced the contrast in type-2b species. Thirdly, separate ¹⁴C-radioassays of vein and mesophyll tissues confirmed this observation. The three-step procedure thus revealed a strong and consistent reduction of phloem loading by PCMBS in type-2b species which was absent in type-1 species. In conclusion, phloem loading in type-2b species occurs via the apoplast and type-1 species execute an alternative — most likely symplasmic — mode of phloem loading.Abbreviations PCMBS p-chloromercuribenzenesulfonic acid - SE/CC-complex sieve element/companion cell complex We gratefully acknowledge the expert help of Dr. Maarten Terlou, Department of Image Processing and Design, University of Utrecht, in carrying out the densitometric measurements.     

Pathway of phloem loading in the C3 tropical orchid hybrid Oncidium Goldiana

The apoplast of mature leaves of the tropical orchid OncidiumGoldiana was perfused with 0.5 mM p-chloromercuribenzenesulphonicacid (PCMBS) via the transpiration stream in order to test themode of phloem loading. The efficacy of introducing PCMBS byperfusion was shown by saffranin O dye movement in the veinsand leaf apoplast in control experiments. Photoassimilate exportas the result of phloem loading was measured by collection of¹⁴CO₂-derived photoassimilates from the basal cut-ends of intactleaves. Phloem loading and translocation of photoassimilates was inhibitedby 89% in leaves perfused with PCMBS for 1 h. The effect ofPCMBS on leaf photosynthesis was minimal. The amount of radiocarbonfixed by PCMBS-treated leaves averaged 89% of control leavesperfused with distilled water. A negative correlation betweenthe total amount of photoassimilate exuded and the calculatedconcentration of PCMBS in the leaf apoplast was also observed.The results indicate that phloem loading in Oncidium Goldianaoccurs via the apoplastic pathway. Key words: Phloem loading, apoplast, PCMBS, tropical orchid     

Sugar synthesis and phloem loading in Coleus blumei leaves

Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of ¹⁴CO₂ was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either ¹⁴CO₂ or [¹⁴C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [¹⁴C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to ¹⁴CO₂ for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of ¹⁴CO₂, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244–1249) indicate that the phloem loading pathway in Coleus is probably symplastic.Abbreviations 3-OMG 3-O-methyl glucose - PCMBS p-chloromercuribenzenesulfonic acid - SE-CCC sieve-element-companion-cell complex This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.     

Ultrastructural indications for coexistence of symplastic and apoplastic phloem loading in Commelina benghalensis leaves

The ultrastructural ontogeny of Commelina benghalensis minor-vein elements was followed. The mature minor vein has a restricted number of elements: a sheath of six to eight mestome cells encloses one xylem vessel, three to five vascular parenchyma cells, a companion cell, a thin-walled protophloem sieve-tube member and a thick-walled metaphloem sieve-tube member. The protophloem sieve-tube member (diameter 4–5 m; wall thickness 0.12 m) and the companion cell originated from a common mother cell. The metaphloem sieve-tube member (diameter 3 m; wall thickness 0.2 m) developed from the same precursor cell as the phloem parenchyma cells. Counting the plasmodesmatal frequencies demonstrated a symplastic continuum from mesophyll to the minor-vein phloem. The metaphloem sievetube member and the phloem parenchyma cells are the termini of this symplast. The protophloem sieve-tube member and companion cell constitute an insulated symplastic domain. The symplastic route, mesophyll to metaphloem sieve tube, appears to offer a path for symplastic loading; the protophloem sieve tube may be capable of accumulation from the apoplast. A similar two-way system of loading may exist in a number of plant families. Plasmodesmograms (a novel way to depict cell elements, plasmodesmatal frequencies and vein architecture) of some other species also displayed the anatomical requirements for two routes from mesophyll to sieve tube and indicate the potential coexistence of symplastic and apoplastic loading.     

Similar photosynthetic response to elevated carbon dioxide concentration in species with different phloem loading strategies

Species have different strategies for loading sugars into the phloem, which vary in the route that sugars take to enter the phloem and the energetics of sugar accumulation. Species with passive phloem loading are hypothesized to have less flexibility in response to changes in some environmental conditions because sucrose export from mesophyll cells is dependent on fixed anatomical plasmodesmatal connections. Passive phloem loaders also have high mesophyll sugar content, and may be less likely to exhibit sugar-mediated down-regulation of photosynthetic capacity at elevated CO₂ concentrations. To date, the effect of phloem loading strategy on the response of plant carbon metabolism to rising atmospheric CO₂ concentrations is unclear, despite the widespread impacts of rising CO₂ on plants. Over three field seasons, five species with apoplastic loading, passive loading, or polymer-trapping were grown at ambient and elevated CO₂ concentration in free air concentration enrichment plots. Light-saturated rate of photosynthesis, photosynthetic capacity, leaf carbohydrate content, and anatomy were measured and compared among the species. All five species showed significant stimulation in midday photosynthetic CO₂ uptake by elevated CO₂ even though the two passive loading species showed significant down-regulation of maximum Rubisco carboxylation capacity at elevated CO₂. There was a trend toward greater starch accumulation at elevated CO₂ in all species, and was most pronounced in passive loaders. From this study, we cannot conclude that phloem loading strategy is a key determinant of plant response to elevated CO₂, but compelling differences in response counter to our hypothesis were observed. A phylogenetically controlled experiment with more species may be needed to fully test the hypothesis.     

Ultrastructure of minor-vein phloem and assimilate export in summer and winter leaves of the symplasmically loading evergreens Ajuga reptans L., Aucuba japonica Thunb., and Hedera helix L.

Minor-vein ultrastructure and sugar export were studied in mature summer and winter leaves of the three broadleaf-evergreen species Ajuga reptans var. artropurpurescens L., Aucuba japonica Thunb. and Hedera helix L. to assess temperature effects on phloem loading. Leaves of the perennial herb Ajuga exported substantial amounts of assimilates in form of raffinose-family oligosaccharides (RFOs). Its minor-vein companion cells represent typical intermediary cells (ICs), with numerous small vacuoles and abundant plasmodesmal connectivity to the bundle sheath. The woody plants Hedera and Aucuba translocated sucrose as the dominant sugar species, and only traces of RFOs. Their minor-vein phloem possessed a layer of highly vacuolated cells (VCs) intervening between mesophyll and sieve elements. Depending on their location and ontogeny, VCs were classified either as companion or parenchyma cells. Both cell types showed symplasmic continuity to the adjacent mesophyll tissue although at a lower plasmodesmal frequency compared to the Ajuga ICs. p-Chloromercuribenzenesulfonic acid did not reduce leaf sugar export in any of the plants, indicating a symplasmic mode of phloem loading. Winter leaves did not show symptoms of frost injury, and the vacuolar pattern in ICs and VCs was equally prominent in both seasons. Starch accumulation as a result of reduced phloem loading was not observed to be triggered by low temperature. In contrast, high amounts of starch were found in mesophyll and bundle-sheath cells of summer leaves. Physiological data on season-dependent leaf exudation showed the maintenance of sugar export in cold-acclimated winter leaves.     

Evidence for an essential role of the sucrose transporter in phloem loading and assimilate partitioning.

Sucrose is the principal transport form of assimilates in most plants. In many species, translocation of assimilates from the mesophyll into the phloem for long distance transport is assumed to be carrier mediated. A putative sucrose proton cotransporter cDNA has been isolated from potato and shown to be expressed mainly in the phloem of mature exporting leaves. To study the in vivo role and function of the protein, potato plants were transformed with an antisense construct of the sucrose transporter cDNA under control of the CaMV 35S promoter. Upon maturation of the leaves, five transformants that expressed reduced levels of sucrose transporter mRNA developed local bleaching and curling of leaves. These leaves contained > 20-fold higher concentrations of soluble carbohydrates and showed a 5-fold increase in starch content as compared with wild type plants, as expected from a block in export. Transgenic plants with a reduced amount of sucrose carrier mRNA show a dramatic reduction in root development and tuber yield. Maximal photosynthetic activity was reduced at least in the strongly affected transformants. The effects observed in the antisense plants strongly support an apoplastic model for phloem loading, in which the sucrose transporter located at the phloem plasma membrane represents the primary route for sugar uptake into the long distance distribution network.     

Functional characterization of the Arabidopsis AtSUC2 Sucrose/H+ symporter by tissue-specific complementation reveals an essential role in phloem loading but not in long-distance transport

AtSUC2 (At1g22710) encodes a phloem-localized sucrose (Suc)/H(+) symporter necessary for efficient Suc transport from source tissues to sink tissues in Arabidopsis (Arabidopsis thaliana). AtSUC2 is highly expressed in the collection phloem of mature leaves, and its function in phloem loading is well established. AtSUC2, however, is also expressed strongly in the transport phloem, where its role is more ambiguous, and it has been implicated in mediating both efflux and retrieval to and from flanking tissues via the apoplast. To characterize the role of AtSUC2 in controlling carbon partitioning along the phloem path, AtSUC2 cDNA was expressed from tissue-specific promoters in an Atsuc2 mutant background. Suc transport in this mutant is highly compromised, as indicated by stunted growth and the accumulation of large quantities of sugar and starch in vegetative tissues. Expression of AtSUC2 cDNA from the 2-kb AtSUC2 promoter was sufficient to restore growth and carbon partitioning to nearly wild-type levels. The GALACTINOL SYNTHASE promoter of Cucumis melo (CmGAS1p) confers expression only in the minor veins of mature leaves, not in the transport phloem of larger leaf veins and stems. Mutant plants expressing AtSUC2 cDNA from CmGAS1p had intermediate growth and accumulated sugar and starch, but otherwise they had normal morphology. These characteristics support a role for AtSUC2 in retrieval but not efflux along the transport phloem and show that the only vital function of AtSUC2 in photoassimilate distribution is phloem loading. In addition, Atsuc2 mutant plants, although debilitated, do grow, and AtSUC2-independent modes of phloem transport are discussed, including an entirely symplastic pathway from mesophyll cells to sink tissues.     

Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in <Emphasis Type="Italic">Plantago major,Plantago maritima</Emphasis>, <Emphasis Type="Italic">Prunus persica</Emphasis>, and <Emphasis Type="Italic">Apium graveolens</Emphasis>

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem.     

PLASMODESMATAL FREQUENCY AND OTHER STRUCTURAL ASPECTS OF ASSIMILATE COLLECTION AND PHLOEM LOADING IN LEAVES OF SONCHUS OLERACEUS (ASTERACEAE), A SPECIES WITH MINOR VEIN TRANSFER CELLS

Leaves of Sonchus oleraceus (Asteraceae) were examined with the electron microscope to determine plasmodesmatal frequencies and other structural features relating to the collection of photoassimilate and its subsequent loading into minor veins. Few plasmodesmata occur between mesophyll cells, which contain chloroplasts that are sometimes connected to both the plasmalemma and the tonoplast by membranous tubules. The minor veins consist of tracheary elements, sieve-tube members, vascular parenchyma cells, and companion cells. The latter two cell types are transfer cells, with some of the fingerlike wall ingrowths in companion cells being traversed lengthwise by plasmodesmata. The frequencies of plasmodesmata at the mesophyllbundle sheath boundary and within are higher at some interfaces than at corresponding interfaces in nine other species, including some that previously had been characterized as loading assimilate via the symplast. It is thus premature to designate all species containing transfer cells in their minor veins as loading assimilate only via the apoplast.     

Evidence for phloem loading via the abaxial bundle sheath cells in maize leaves

Leaves are asymmetric, with different functions for adaxial and abaxial tissue. The bundle sheath (BS) of C₃ barley (Hordeum vulgare) is dorsoventrally differentiated into three types of cells: adaxial structural, lateral S-type, and abaxial L-type BS cells. Based on plasmodesmatal connections between S-type cells and mestome sheath (parenchymatous cell layer below bundle sheath), S-type cells likely transfer assimilates toward the phloem. Here, we used single-cell RNA sequencing to investigate BS differentiation in C₄ maize (Zea mays L.) plants. Abaxial BS (^abBS) cells of rank-2 intermediate veins specifically expressed three SWEET sucrose uniporters (SWEET13a, b, and c) and UmamiT amino acid efflux transporters. SWEET13a, b, c mRNAs were also detected in the phloem parenchyma (PP). We show that maize has acquired a mechanism for phloem loading in which ^abBS cells provide the main route for apoplasmic sucrose transfer toward the phloem. This putative route predominates in veins responsible for phloem loading (rank-2 intermediate), whereas rank-1 intermediate and major veins export sucrose from the PP adjacent to the sieve element companion cell complex, as in Arabidopsis thaliana. We surmise that ^abBS identity is subject to dorsoventral patterning and has components of PP identity. These observations provide insights into the unique transport-specific properties of ^abBS cells and support a modification to the canonical phloem loading pathway in maize.     

Immunolocalization of solanaceous SUT1 proteins in companion cells and xylem parenchyma: new perspectives for phloem loading and transport

Leaf sucrose (Suc) transporters are essential for phloem loading and long-distance partitioning of assimilates in plants that load their phloem from the apoplast. Suc loading into the phloem is indispensable for the generation of the osmotic potential difference that drives phloem bulk flow and is central for the long-distance movement of phloem sap compounds, including hormones and signaling molecules. In previous analyses, solanaceous SUT1 Suc transporters from tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and tomato (Solanum lycopersicum) were immunolocalized in plasma membranes of enucleate sieve elements. Here, we present data that identify solanaceous SUT1 proteins with high specificity in phloem companion cells. Moreover, comparisons of SUT1 localization in the abaxial and adaxial phloem revealed higher levels of SUT1 protein in the abaxial phloem of all three solanaceous species, suggesting different physiological roles for these two types of phloem. Finally, SUT1 proteins were identified in files of xylem parenchyma cells, mainly in the bicollateral veins. Together, our data provide new insight into the role of SUT1 proteins in solanaceous species.     

The evolution of minor vein phloem and phloem loading

Phylogenetic analysis provides a rational basis for comparative studies of phloem structure and phloem loading. Although several types of minor vein companion cell have been identified, and progress has been made in correlating structural features of these cells with loading mechanisms, little is known about the phylogenetic relationships of the different types. To add to the available data on companion cells, we analyzed the ultrastructure of minor veins in Euonymus fortunei and Celastrus orbiculatis (Celastraceae) leaves and determined that in these species they are specialized as intermediary cells. This cell type has been implicated in symplastic phloem loading. The data were added to published data sets on minor vein phloem characteristics, which were then mapped to a well-supported molecular tree. The analysis indicates that extensive plasmodesmatal continuity between minor vein phloem and surrounding cells is ancestral in the angiosperms. Reduction in plasmodesmatal frequency at this interface is a general evolutionary trend, punctuated by instances of the reverse. This is especially true in the case of intermediary cells that have many plasmodesmata, but other distinguishing characteristics as well, and have arisen independently at least four, and probably six, times in derived lineages. The character of highly reduced plasmodesmatal frequency in minor vein phloem, common in crop plants, has several points of origin in the tree. Thus, caution should be exercised in generalizing results on apoplastic phloem loading obtained from model species. Transfer cells have many independent points of origin, not always from lineages with reduced plasmodesmatal frequency.     

Symplasmic transport and phloem loading in gymnosperm leaves

Despite more than 130 years of research, phloem loading is far from being understood in gymnosperms. In part this is due to the special architecture of their leaves. They differ from angiosperm leaves among others by having a transfusion tissue between bundle sheath and the axial vascular elements. This article reviews the somewhat inaccessible and/or neglected literature and identifies the key points for pre-phloem transport and loading of photoassimilates. The pre-phloem pathway of assimilates is structurally characterized by a high number of plasmodesmata between all cell types starting in the mesophyll and continuing via bundle sheath, transfusion parenchyma, Strasburger cells up to the sieve elements. Occurrence of median cavities and branching indicates that primary plasmodesmata get secondarily modified and multiplied during expansion growth. Only functional tests can elucidate whether this symplasmic pathway is indeed continuous for assimilates, and if phloem loading in gymnosperms is comparable with the symplasmic loading mode in many angiosperm trees. In contrast to angiosperms, the bundle sheath has properties of an endodermis and is equipped with Casparian strips or other wall modifications that form a domain border for any apoplasmic transport. It constitutes a key point of control for nutrient transport, where the opposing flow of mineral nutrients and photoassimilates has to be accommodated in each single cell, bringing to mind the principle of a revolving door. The review lists a number of experiments needed to elucidate the mode of phloem loading in gymnosperms.     

Long-distance trafficking of macromolecules in the phloem

The fact that macromolecules such as proteins and mRNAs overcome the symplastic barriers between various tissue domains was first evidenced by the movement of plant viruses. We have recently demonstrated that viral infection disengages the symplastic restriction present between the sieve element-companion cell complex and neighboring cells in tobacco plants. As a result, green fluorescent protein, which was produced in mesophyll and bundle sheath cells, could traffic into the sieve tube and travel long distances within the vascular system. In this addendum we discuss the likely existence of a novel plant communication network in which macromolecules also act as long-distance trafficking signals. Plasmodesmata interconnecting sieve elements and companion cells as well as plasmodesmata connecting the sieve tube with neighboring cells may play a central role in establishing this communication network.Key words: companion cells, cucumber mosaic virus, Cucumis melo, plasmodesmata, movement protein, sieve-elementsTranslocation of photoassimilates from the source (site of synthesis) to various sink organs is governed, in part, by short-distance intercellular transfer of assimilates to the loading region of the phloem and long-distance transport within the plant vascular system. Sucrose, which is synthesized in the leaf mesophyll, moves cell-to-cell symplastically through plasmodesmata until it reaches the boundary of the sieve element (SE)-companion cell (CC) complex. In many plant species, the connection between phloem parenchyma (PP)/bundle sheath (BS) cells and CCs is characterized by a sparseness of plasmodesmata (e.g., Solanaceae), and sucrose is exported out of the cells to the apoplast. This type of plants (apoplastic loaders) uses sucrose proton symporters to load the sucrose into the vasculature.¹ Cucurbits are considered one of the model plants for symplastic phloem loading.² This type of plant is characterized by abundant plasmodesmata interconnecting the intermediary cells, which are specialized CCs, with the neighboring BS cells. It is generally accepted that in these plants, phloem loading includes intercellular movement of sucrose through the plasmodesmata, along the entire pathway from the mesophyll cell to the SE-CC complex.Interestingly, the existence of plasmodesmata interconnecting the SE-CC complex and neighboring cells is evident in all plant species that are characterized by an apoplastic phloem-loading mechanism. Moreover, microinjection experiments have indicated that plasmodesmata interconnecting the PP-CC are functional, in that they allow the exchange of small membrane-impermeable fluorescent probes.³ Virus movement through plasmodesmata from the mesophyll into the SEs further supports the notion that the symplastic communication between the CC-SE complex and the neighboring cells is functional.⁴One can assume that in apoplastic-loading plants, it would be an advantage to maintain the SE-CC complex as an isolated domain, with no functional plasmodesmata interconnecting it to the neighboring tissue. Symplastic continuity between the two domains could result in leakage of sucrose out of the vasculature and a significant reduction in the efficacy of sucrose loading. The fact that the two domains are interconnected suggests that any back-leakage of sucrose that might occur is insignificant relative to the likely efficacy of this communication route.What might the advantage be for symplastic communication between the SE-CC complex and the neighboring tissue? Accumulated evidence suggests that at the tissue/organ level, cell-to-cell trafficking of information molecules allows for noncell-autonomous control over a range of processes, whereas at the organismal level, the phloem serves as an information superhighway, delivering a wide range of macromolecules to enable the plant to function as a whole organism.^5–8 We advanced the hypothesis that plasmodesmata interconnecting the CCs and PP/BS cells play a pivotal role in controlling the long-distance trafficking of putative signaling molecules.     

Viral infection enables phloem loading of GFP and long-distance trafficking of the protein

It is generally accepted that viral systemic infection follows the source-to-sink symplastic pathway of sugar translocation. In plants that are classified as apoplastic loaders, the boundary between the companion cell-sieve element (CC-SE) complex and neighboring cells is symplastically restricted, and the potential passage of macromolecules between the two domains has yet to be explored. Transgenic tobacco plants expressing green fluorescence protein (GFP) and cucumber mosaic virus (CMV)-encoded proteins fused to GFP under the control of the fructose-1,6-bisphosphatase (FBPase) promoter were produced in order to localize the encoded proteins in mesophyll and bundle sheath cells and to explore the influence of viral infection on the functioning of plasmodesmata interconnecting the two domains. GFP produced outside the vascular tissue could overcome the symplastic barrier between the CC-SE complex and the surrounding cells to enter the vasculature in CMV-infected plants. Grafting of control (non-transgenic) tobacco scions to CMV-infected FBPase-GFP-expressing root stocks confirmed that GFP could move long distances in the phloem. No movement of the gfp mRNA was noticeable in this set of experiments. The ability of GFP to enter the vasculature and move long distances was also evident upon infection of the grafting plants with other viruses. These results provide experimental evidence for alteration of the functioning of plasmodesmata interconnecting the CC-SE complex and neighboring cells by viral infection to enable non-selective trafficking of macromolecules from the mesophyll into the sieve tube.